Figure 3. ATG5 affects sorting of the retromer cargo GLUT1.

(A) Coimmunoprecipitation (co-IP) analysis of endogenous ATG5 with GFP-VPS35 or YFP-VPS29 (transient transfection). (B) Reverse co-IP analysis GFP-VPS35 or YFP-VPS29 (transient transfection) and endogenous ATG5. Note that different antibodies were used in panels A and B: ab108327 (Abcam) recognizing preferentially the conjugated form of ATG5 and abASA-B0113 (Novateinbio), recognizing both conjugated and unconjugated ATG5. (C) Confocal images illustrating localization of GLUT1 and LAMP2 in Huh7^WT, Huh7^VPS35-KO, and Huh7^ATG5-KO cells. Scale bar, 10 μm. High content microscopy (HCM) quantification of GLUT1 (endogenous protein immunostaining) puncta/cell (D) and GLUT1 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) (E) in Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 6); one-way ANOVA with Tukey’s multiple comparisons. (F, G) HCM quantification of GLUT1-SNX27 overlap (% of SNX7 area positive for GLUT1) in Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (H, I) Immunoblot analysis and quantification of proteins in lysosomes purified/enriched by LysoIP (immunoisolation with TMEM192-3xHA) from Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. TMEM192-2xFLAG, negative control. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. HCM images in panel F, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line.

Figure 3—figure supplement 1. Membrane atg8ylation regulates retromer function.

(A) Immunoblots of CRISPR KOs: (i) ATG3, ATG5, and ATG7 in Huh7 cells; (ii) VPS35 in Huh7 cells; (iii) ATG5 in HeLa cells; and (iv) VPS35 in Hela cells. High content microscopy (HCM) images (example form a bank of unbiased operator-independent machine-collected and processed images containing a minimum of 500 primary objects/cells) of GLUT1 (immunostaining of endogenous protein) puncta/cell (B) and GLUT1 colocalization with LAMP2 in Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells (C). Scale bar, 20 μm. (D, E) HCM quantification of GLUT1 (immunostaining of endogenous protein) puncta/cell in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (F, G) HCM quantification of GLUT1 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (H) Confocal images illustrating localization of GLUT1 and LAMP2 in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells. Scale bar, 10 μm. (I, J) HCM quantification of GLUT1-SNX27 overlap (% of SNX7 area positive for GLUT1) in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO. Scale bar, 20 μm. Data, means ± SE (n = 4); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line.